Anandamide Uptake Is Consistent with Rate-limited Diffusion and Is Regulated by the Degree of Its Hydrolysis by Fatty Acid Amide Hydrolase

Abstract

The uptake of arachidonoyl ethanolamide (anandamide, AEA) in rat basophilic leukemia cells (RBL-2H3) has been proposed to occur via a saturable transporter that is blocked by specific inhibitors. Measuring uptake at 25 s, when fatty acid amide hydrolase (FAAH) does not appreciably affect uptake, AEA accumulated via a nonsaturable mechanism at 37°C. Interestingly, saturation was observed when uptake was plotted using unbound AEA at 37°C. Such apparent saturation can be explained by rate-limited delivery of AEA through an unstirred water layer surrounding the cells (1). In support of this, we observed kinetics consistent with rate-limited diffusion at 0°C. Novel transport inhibitors have been synthesized that are either weak FAAH inhibitors or do not inhibit FAAH in vitro (e.g. UCM707, OMDM2, and AM1172). In the current study, none of these purported AEA transporter inhibitors affected uptake at 25 s. Longer incubation times illuminate downstream events that drive AEA uptake. Unlike the situation at 25 s, the efficacy of these inhibitors was unmasked at 5 min with appreciable inhibition of AEA accumulation correlating with partial inhibition of AEA hydrolysis. The uptake and hydrolysis profiles observed with UCM707, VDM11, OMDM2, and AM1172 mirrored two selective and potent FAAH inhibitors CAY10400 and URB597 (at low concentrations), indicating that weak inhibition of FAAH can have a pronounced effect upon AEA uptake. At 5 min, the putative transport inhibitors did not reduce AEA uptake in FAAH chemical knock-out cells. This strongly suggests that the target of UCM707, VDM11, OMDM2, and AM1172 is not a transporter at the plasma membrane but rather FAAH, or an uncharacterized intracellular component that delivers AEA to FAAH. This system is therefore unique among neuro/immune modulators because AEA, an uncharged hydrophobic molecule, diffuses into cells and partial inhibition of FAAH has a pronounced effect upon its uptake. Arachidonoyl ethanolamide (AEA)2 is a neuromodulatory lipid that belongs to a family of molecules collectively termed the endocannabinoids. Like ⌬ 9 -tetrahydracannabinol, many of the actions of AEA are mediated through the G-protein-coupled cannabinoid (CB 1 and CB 2 ) and vanilloid (TRPV1) receptors (2-7). AEA signaling is terminated by a rapid reuptake mechanism followed by its hydrolysis into arachidonic acid and ethanolamine primarily by the intracellular enzyme FAAH (8 -10). By metabolizing AEA, FAAH maintains an inward gradient that drives the continued cellular accumulation of AEA (11-13).There is some controversy regarding the processes mediating AEA uptake. It was widely accepted that AEA transport is a selective, saturable process of facilitated diffusion for which there are selective transport inhibitors (For review, see Refs. 14 -16). However, in platelets, neuroblastoma, astrocytoma, primary cortical neurons, and erythrocyte ghost membranes it has been suggested that uptake occurs by passive diffusion (13,(17)(18)(19)(20). Experimental variables such as incubation times, inclusion ...

Identification of intracellular carriers for the endocannabinoid anandamide

“…Although many of these structural analogs of AEA reduce its uptake by inhibiting FAAH (11,12,58,59), several compounds have recently been described that do not appreciably inhibit this enzyme (60,61). Several groups have hypothesized these compounds may act upon a putative AEA membrane transporter; however, it has also been speculated that these compounds inhibit intracellular trafficking proteins (11,12,14,23).…”

“…These assays tracked the cellular uptake of [ 14 C]AEA and its subsequent breakdown into [ 14 C]ethanolamine by FAAH (12). AEA uptake is coupled to its hydrolysis by FAAH, which maintains an outward/inward concentration gradient that increases AEA accumulation in the steady state (12,39,40). Therefore, if AEA delivery to FAAH is reduced when FAAH expression is confined to the Golgi apparatus or mitochondria, the reduced hydrolysis of AEA will lower the AEA gradient and diminish its uptake.…”

“…FAAH activity assays were performed as previously described (12). Briefly, cell homogenates were incubated with 100 M AEA ϩ 0.…”

“…In contrast, AEA is an uncharged lipid capable of traversing the bilayer unaided (10). Accordingly, several studies concluded that AEA uptake occurs by passive diffusion (10)(11)(12)(13)(14)(15), although facilitated diffusion and/or endocytosis have also been proposed (16)(17)(18)(19)(20).…”

Design, Synthesis, and In Vitro Evaluation of Carbamate Derivatives of 2-Benzoxazolyl- and 2-Benzothiazolyl-(3-hydroxyphenyl)-methanones as Novel Fatty Acid Amide Hydrolase Inhibitors

“…22 In addition, inhibitors have been developed for the transport protein, 12,23,24 although its existence is under debate. 12,25,26 The enzyme inhibition could be a convenient way to elevate the endocannabinoid levels and thus increase the receptor activity. 27 Endocannabinoids are biosynthesized upon demand and released immediately from neurons afterward.…”